JPH0228098B2 - ENDOTOKISHINKENSHUTSUHO - Google Patents
ENDOTOKISHINKENSHUTSUHOInfo
- Publication number
- JPH0228098B2 JPH0228098B2 JP2296384A JP2296384A JPH0228098B2 JP H0228098 B2 JPH0228098 B2 JP H0228098B2 JP 2296384 A JP2296384 A JP 2296384A JP 2296384 A JP2296384 A JP 2296384A JP H0228098 B2 JPH0228098 B2 JP H0228098B2
- Authority
- JP
- Japan
- Prior art keywords
- endotoxin
- tube
- liquid
- test
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 239000002158 endotoxin Substances 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 14
- 241001529572 Chaceon affinis Species 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 239000012085 test solution Substances 0.000 claims description 11
- 210000000601 blood cell Anatomy 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 5
- 210000005239 tubule Anatomy 0.000 claims description 2
- 230000000717 retained effect Effects 0.000 claims 1
- 239000006166 lysate Substances 0.000 description 11
- 238000001879 gelation Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000010998 test method Methods 0.000 description 5
- 241000239218 Limulus Species 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000011109 contamination Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000003677 hemocyte Anatomy 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- 239000000815 hypotonic solution Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 239000002683 reaction inhibitor Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
【発明の詳細な説明】
(イ) 技術分野
本発明はエンドトキシン検出法に関する。さら
に詳しくは、リムルステスト法におけるゲル化反
応の測定法に関する。DETAILED DESCRIPTION OF THE INVENTION (a) Technical field The present invention relates to a method for detecting endotoxin. More specifically, the present invention relates to a method for measuring gelation reaction in the Limulus test method.
(ロ) 従来技術
エンドトキシンはグラム陰性菌の細胞壁中に含
まれるリポ多糖と云われており、熱に対してかな
り安定であり、ヒトに対して発熱作用が著しく
0.1μg程度の微量でも血管内に入ると発熱を生じ、
多量の場合は組織の破壊、シヨツク死等を起こす
と云われている。近年特に、エンドトキシン汚染
による透析液の副作用は大きな問題になつてい
る。エンドトキシンの微量検出法として、従来ウ
サギ発熱性試験等のin vivoでの方法が用いられ
てきたが、最近in vitroで試験できるリムルステ
スト法が開発された。このテスト法はカブトガニ
の血球抽出液（以下「ライセート」と略す）のゲ
ル化反応を利用するもので、従来法に比較し検出
時間が短く、しかも検出感度も高い。従つて、医
療器具、注射剤等の製造工程管理に用いられつつ
ある。また、血液の診断、食品中の細菌汚染の判
定等に用いる試みもなされている。(b) Prior art Endotoxin is said to be a lipopolysaccharide contained in the cell wall of Gram-negative bacteria, and is quite stable against heat, and has a significant fever effect on humans.
Even a minute amount of about 0.1μg can cause fever if it enters the bloodstream.
In large amounts, it is said to cause tissue destruction and shock death. In recent years, side effects of dialysate fluids due to endotoxin contamination have become a major problem. Conventionally, in vivo methods such as the rabbit pyrogenicity test have been used to detect trace amounts of endotoxin, but recently the Limulus test method, which can be tested in vitro, has been developed. This test method utilizes the gelation reaction of horseshoe crab blood cell extract (hereinafter abbreviated as "lysate"), and has a shorter detection time and higher detection sensitivity than conventional methods. Therefore, it is being used for manufacturing process control of medical devices, injections, etc. Attempts have also been made to use it for blood diagnosis, determination of bacterial contamination in food, etc.
リムルステスト法にはそのゲル化反応を測定す
る方法として、試験管またはアンプル等の反応容
器内で生じたゲルの崩れ易さを容器を傾けて肉眼
で判定する試験管法、ゲル形成に伴つて濁度の増
加を光度計で測る比濁法、不溶性蛋白質の量を定
量して測定するclot蛋白法等が知られている。こ
れらのリムルステスト法は簡便でありもつとも普
及している方法である。 The limulus test method includes two methods for measuring the gelation reaction: the test tube method, in which the ease with which the gel formed in a reaction container such as a test tube or ampoule collapses is judged with the naked eye by tilting the container; The nephelometric method, which measures the increase in protein density using a photometer, and the clot protein method, which measures the amount of insoluble protein by quantifying it, are known. These Limulus test methods are simple and popular methods.
しかし、これ等の方法は高価なライセートを多
量に用いなければならず検出費用が嵩み、また被
検液も比較的多量に用いなければならない等の欠
点等がある。特にカブトガニ資源の保護のために
も使用ライセートの微量化が望まれている。 However, these methods have drawbacks such as requiring the use of a large amount of expensive lysate, increasing detection costs, and requiring the use of a relatively large amount of test liquid. In particular, it is desired to reduce the amount of lysate used in order to protect horseshoe crab resources.
(ハ) 発明の目的
本発明の目的は、微量のライセートと被検液を
用いて、エンドトキシンを従来法に比較し鋭敏に
検出測定できる方法を提供するにある。(c) Purpose of the Invention The purpose of the present invention is to provide a method that can detect and measure endotoxin more sensitively than conventional methods using a small amount of lysate and test solution.
(ニ) 発明の構成
本発明に係るエンドトキシンの検出法は、エン
ドトキシンを含有する被検液とカブトガニの血球
抽出成分とを混和した液を透明細管内に保持し、
一定温度で反応せしめた後該細管を傾けて液の移
動の有無によりエンドトキシン濃度の測定を行な
うことを特徴とする。(d) Structure of the Invention The method for detecting endotoxin according to the present invention involves holding a mixture of a test solution containing endotoxin and a horseshoe crab blood cell extract in a transparent tubule;
The method is characterized in that after reacting at a constant temperature, the tube is tilted and the endotoxin concentration is measured based on the presence or absence of liquid movement.
(ホ) 発明の効果
本発明はエンドトキシンとライセートを含む液
が細管内でゲル化すると管内を移動しなくなると
いう知見に基づいて完成されたものであつて、従
来法に比較して、検出感度が高く、しかもライセ
ートおよび被検液の使用量が少なくてすむ点で優
れている。(e) Effects of the invention The present invention was completed based on the knowledge that when a liquid containing endotoxin and lysate gels in a capillary, it stops moving within the tube, and the detection sensitivity is higher than that of conventional methods. It is superior in that it is expensive and requires less amount of lysate and test solution.
(ヘ) 発明の構成の具体的説明
本発明で使用するのカブトガニ血球抽出成分は
通常、カブトガニの血球中に含まれるアメーバー
様細胞から低張液で抽出処理して得られる抽出
液、または、この抽出液の凍結乾燥物であるが、
抽出成分にエンドトキシンと反応してゲル化を起
こすものが含まれていれば、その形態は格別限定
されない。また、抽出成分に牛血清アルブミン等
の成分安定化剤あるいはCa++、Mg++等の活性化
剤を添加したものを用いてもさしつかえない。(f) Specific description of the composition of the invention The horseshoe crab blood cell extract component used in the present invention is usually an extract obtained by extracting ameboid cells contained in horseshoe crab blood cells with a hypotonic solution, or It is a freeze-dried product of the extract,
The form is not particularly limited as long as the extracted component contains something that reacts with endotoxin and causes gelation. Further, it is also possible to use an extracted component to which a component stabilizer such as bovine serum albumin or an activator such as Ca ++ or Mg ++ is added.
エンドトキシンを含有する被検液は、エンドト
キシンが溶解したあるいはエンドトキシンを有す
る細菌が懸濁した水溶液であり、蛋白変性剤、界
面活剤性等のゲル化反応阻害剤を含まないものを
云う。また、液のPHが６〜８であるものを使用す
る。 The test solution containing endotoxin is an aqueous solution in which endotoxin is dissolved or endotoxin-containing bacteria are suspended, and does not contain gelation reaction inhibitors such as protein denaturants and surfactants. Also, use a solution whose pH is 6 to 8.
被検液にライセートを溶解して得られる混和液
は、細管内で調整した液でもあるいは細管外で調
整のした液でもかまわない。予め細管内にライセ
ートの乾燥物を保持しついで被検液を導入して溶
解する方法によつて調製した混和液は、その量が
少なくてすみまた操作中の汚染を防ぐことができ
る点で都合がよい。また、混和液中のライセート
の濃度はエンドトキシンにより液がゲル化し得る
程度であればよく、試験管法で用いられている濃
度と同程度のものが用いらる。透明細管としては
水平に静置した時液を管内壁にに沿つて保持し、
管内の液が容易に流出しない程度の内径を有する
ものであればよく、好ましくは内径0.1〜５mmで
ある。また、その材質は管内に存在する液の状態
が観察できる程度の透明性を有するものであれば
無機質でも有機質でもよい。例えば、透明細管は
ガラス、ポリメチルメタクリレート、ポリスチレ
ン等である。 The mixed solution obtained by dissolving the lysate in the test solution may be a solution prepared inside the capillary or a solution prepared outside the capillary. A mixed solution prepared by holding the dry lysate in a capillary in advance and introducing and dissolving the test solution is convenient because the amount required is small and contamination during operation can be prevented. Good. Further, the concentration of lysate in the mixed solution is sufficient as long as the solution can be gelled by endotoxin, and the concentration used is about the same as that used in the test tube method. As a transparent thin tube, when it is placed horizontally, it retains the liquid along the inner wall of the tube.
The tube may have an inner diameter that does not allow the liquid inside the tube to easily flow out, and preferably has an inner diameter of 0.1 to 5 mm. Further, the material may be inorganic or organic as long as it has transparency to the extent that the state of the liquid present in the tube can be observed. For example, the transparent capillary is made of glass, polymethyl methacrylate, polystyrene, or the like.
混和液を「透明細管内に保持する」とは液を管
内壁に沿つて静止した状態で存在させることであ
り、その管内における液長は0.1〜３cm程度が好
ましい。液長を長くすることは混和液を多量に必
要とすることを意味し、ひいてはライセートおよ
び被検液を多量に必要とする。また、短すぎると
再現性良く検出することが難かしくなる。 "Holding the mixed liquid in a transparent capillary" means that the liquid exists in a stationary state along the inner wall of the tube, and the length of the liquid in the tube is preferably about 0.1 to 3 cm. Increasing the liquid length means requiring a large amount of mixed solution, which in turn requires large amounts of lysate and test solution. Moreover, if it is too short, it will be difficult to detect with good reproducibility.
次に、「一定温度で反応せしめる」とは一定の
温度に保ちながらライセートとエンドトキシンと
のゲル化反応を行なうことである。その温度は反
応が進行する温度であればよいが、最適な反応温
度であれば反応時間も短く最も好都合である。通
常、最適温度は約37℃であり、この温度に保たれ
た恒温槽に細管を水平に静止することで行なわれ
る。「細管を傾けて反応液の移動の有無を判定す
る」とは、反応後、細管をほぼ垂直にし反応液が
管壁に沿つて落下するか否かによりエンドトキシ
ンの濃度を判定することである。落下しない時は
所定量のエンドトキシンが被検液中に含まれ、ま
た、落下した時は所定量のエンドトキシンが含ま
れていないことを意味する。 Next, "reacting at a constant temperature" means performing a gelation reaction between the lysate and endotoxin while maintaining the temperature at a constant temperature. The temperature may be any temperature that allows the reaction to proceed, but an optimal reaction temperature is most convenient because the reaction time is short. Usually, the optimum temperature is about 37°C, and the process is carried out by placing the tube horizontally in a constant temperature bath maintained at this temperature. "Tilting the tube to determine whether the reaction liquid has moved" means that after the reaction, the tube is made almost vertical and the concentration of endotoxin is determined by whether or not the reaction liquid falls along the tube wall. When the test liquid does not fall, a predetermined amount of endotoxin is contained in the test liquid, and when it falls, it means that the predetermined amount of endotoxin is not contained in the test liquid.
(ト) 実施例
以下、実施例について本発明の検出法を具体的
に説明する。(G) Examples Hereinafter, the detection method of the present invention will be specifically explained with reference to Examples.
実施例 １
カブトガニの血球抽出物の凍結乾燥物（製造元
Associate of Cape Cod社、販売元和光純薬工
業）240mgを局方注射用蒸留水５mlに溶解する。
E.coli UKT−Ｂ株起源のエンドトキシン（販売
元和光純薬工業）500ngを局方注射用蒸留水５ml
に溶解する。さらにこれを1000倍に希釈して、
0.07ng／mlの被検液を調整した。カブトガニの血
球抽出物溶解液20μと被検液20μとを混合し、
直にエンドトキシンフリーのガラス管（内径1.6
mm、肉厚0.8mm、管長12cm）にこの混合液を移液、
しさらに管中央部まで液を移動し、この管を37℃
のふらん器内に水平に静置し反応を行なつた。
1.0時間後ふらん器内より取り出し、室温で管を
垂直に立てたが、反応液は静止して落下は見られ
なかつた。Example 1 Freeze-dried horseshoe crab hemocyte extract (manufacturer
Associate of Cape Cod, sold by Wako Pure Chemical Industries) Dissolve 240 mg in 5 ml of distilled water for pharmacopoeial injection.
Add 500 ng of endotoxin originating from E. coli UKT-B strain (sold by Wako Pure Chemical Industries) to 5 ml of distilled water for pharmacopoeial injection.
dissolve in Furthermore, dilute this 1000 times,
A test solution of 0.07 ng/ml was prepared. Mix 20μ of horseshoe crab blood cell extract solution and 20μ of test solution,
Directly into endotoxin-free glass tubing (inner diameter 1.6
Transfer this mixture to a tube (wall thickness: 0.8 mm, tube length: 12 cm).
Then move the liquid to the center of the tube and heat the tube to 37℃.
The reaction was carried out by placing it horizontally in a flannel.
After 1.0 hour, the tube was taken out from the flask and stood vertically at room temperature, but the reaction solution remained stationary and no falling was observed.
一方、被検液として局方注射用蒸留水を用いて
同様な操を行なつた。この場合には反応液は静止
せず、管壁に沿つて急速に落下した。 On the other hand, a similar operation was carried out using distilled water for pharmacopoeial injection as the test liquid. In this case, the reaction solution did not stand still, but rapidly fell along the tube wall.
実施例 ２
実施例１で用いたカブトガニの血球抽出物溶解
液12.5μを実施例１と同じガラス管に導入し、
さらにその液を管中央部に移動し凍結乾燥を行な
つた。このガラス管に実施例１と同じ0.07ng／ml
の被検液を25μ導入して凍結乾燥物を溶解し、
以降実施例１と同様な操作を行なつた。Example 2 12.5μ of the horseshoe crab blood cell extract solution used in Example 1 was introduced into the same glass tube as in Example 1,
Further, the liquid was transferred to the center of the tube and freeze-dried. The same 0.07ng/ml as in Example 1 was added to this glass tube.
Introduce 25μ of the test solution to dissolve the lyophilized product,
Thereafter, the same operations as in Example 1 were performed.
反応液の落下は見られなかつた。また被検液と
して局方注射用蒸留水を用いて同様な操作を行な
つた。この場合反応液は急速に落下した。 No falling of the reaction solution was observed. Further, similar operations were performed using distilled water for pharmacopoeial injection as the test liquid. In this case, the reaction solution dropped rapidly.
Claims (1)
ニの血球抽出成分とを混和した液を透明細管内に
保持し、一定温度で反応せしめた後該細管を傾け
て反応液の移動の有無を判定することことを特徴
とするエンドトキシンの検出法。 ２ カブトガニの血球抽出成分として凍結乾燥品
を用いる特許請求の範囲第１項記載のエンドトキ
シン検出法。 ３ カブトガニの血球抽出成分と被検液との混和
を透明細管内で行なう特許請求範囲第１項または
第２項記載のエンドトキシン検出法。 ４ 透明細管として内径が0.1〜５mmのものを用
いる特許請求範囲第１項、第２項または第３項記
載のエンドトキシン検出法。 ５ 混和した液の透明細管内保持量が液長で表し
た時0.1〜３cmであることを特徴とする特許請求
範囲第１項から第４項までのいずれかに記載のエ
ンドトキシン検出法。[Claims] 1. A mixture of a test solution containing endotoxin and a horseshoe crab blood cell extract is held in a transparent capillary, and after reacting at a constant temperature, the capillary is tilted to control the movement of the reaction solution. A method for detecting endotoxin, characterized by determining the presence or absence of endotoxin. 2. The endotoxin detection method according to claim 1, which uses a freeze-dried product as the horseshoe crab blood cell extract component. 3. The endotoxin detection method according to claim 1 or 2, wherein the horseshoe crab blood cell extract and the test liquid are mixed in a transparent tubule. 4. The endotoxin detection method according to claim 1, 2 or 3, using a transparent capillary having an inner diameter of 0.1 to 5 mm. 5. The endotoxin detection method according to any one of claims 1 to 4, wherein the amount of the mixed liquid retained in the transparent capillary is 0.1 to 3 cm when expressed in liquid length.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2296384A JPH0228098B2 (en) | 1984-02-13 | 1984-02-13 | ENDOTOKISHINKENSHUTSUHO |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2296384A JPH0228098B2 (en) | 1984-02-13 | 1984-02-13 | ENDOTOKISHINKENSHUTSUHO |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60168051A JPS60168051A (en) | 1985-08-31 |
| JPH0228098B2 true JPH0228098B2 (en) | 1990-06-21 |
Family
ID=12097237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2296384A Expired - Lifetime JPH0228098B2 (en) | 1984-02-13 | 1984-02-13 | ENDOTOKISHINKENSHUTSUHO |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0228098B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10793327B2 (en) | 2017-10-09 | 2020-10-06 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| CA3130700A1 (en) | 2019-03-14 | 2020-09-17 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
-
1984
- 1984-02-13 JP JP2296384A patent/JPH0228098B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60168051A (en) | 1985-08-31 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |